Project Summary/Abstract With every breath, the lung is exposed to an array of environmental agents, which must be discriminated as harmful or harmless by the local mononuclear phagocytes (MPs). Dendritic cells (DC) in the lung, part of the MP system, are key to this recognition and to the initiation of immune responses to foreign antigens. However, our current knowledge of the types, functions and locations of DCs in the human lung is extremely limited, and based largely on analogies with more detailed studies in mice. A direct understanding of human DC signaling is a critical step in developing human immune-based therapeutics. In this grant, we propose a detailed functional characterization of human pulmonary DCs. We hypothesize that we will be able to identify two clearly differentiated types of DCs (called here hDC1 and hDC2) playing separate functional roles. Based on mouse models developed in our lab and others', we expect hDC1 and hDC2 populations to have functional characteristics associated with antiviral and antifungal immunity, respectively, though hDCs may also be quite different from mouse DCs in function. More specifically, we will test the hypotheses that hDC1 selectively phagocytose dying cells, express and are activated through Toll-like receptor 3 (TLR3), cross-present cell-associated antigen, and promote the induction of cytotoxic CD8+ T cells. By contrast, hDC2 are expected to be incapable of ingesting apoptotic cells, will express different TLRs (e.g. TLR7) and C-type lectins such as Mincle and Dectin1, and will predominantly present soluble and particulate exogenous antigen to CD4+ T cells. Aim 1 is to investigate the selective acquisition of apoptotic cells (efferocytosis) by human pulmonary DCs. The physiological importance of knowing the efferocytic DC (i.e. cross-presenting DC) has been clearly outlined for viral immunity, intracellular pathogens, and cancer. In Aim 2 we examine gene and protein expression by the proposed human pulmonary DCs, including pattern recognition receptors (PRR) and responses to their ligation. Since antigen-bearing DCs need to be directly stimulated by their cognate PRR to drive the differentiation of effector T cells, characterizing the PRRs, specifically TLRs, expressed by human pulmonary DCs will provide key information for successful activation of target pulmonary DCs in induction of adaptive immune responses. Finally, in Aim 3, we will test the hypothesis that the two candidate human lung DCs differ in their ability to present exogenous antigens, resulting in the differential induction of CD4 and CD8 T cells. The net outcome of these studies will be a detailed functional analysis of the human pulmonary migratory DC populations ? as complete as can be achieved without true in vivo studies, but making optimal use of non-diseased human lungs, along with their attached draining lymph nodes. These studies will heighten our understanding of the human pulmonary immune system and enhance our ability to develop targeted immune therapies for multiple diseases such as lung cancer and COPD.